1. Field of the Invention
The present invention relates to a sensing device and a sensing method for recognizing a substance to be sensed contained in a sample solution and determining its quantity based on a frequency of a piezoelectric resonator such as a quartz-crystal resonator.
2. Description of the Related Art
As a device detecting a trace amount of a substance contained in a sample solution, there has been known a quartz-crystal sensor utilizing a quartz-crystal resonator, and a detection principle of such a quartz-crystal sensor is that an oscillation frequency (resonance frequency) of the quartz-crystal resonator changes when it adsorbs a trace amount of a substance. For example, in such a quartz-crystal sensor, an adsorption layer made of a biological substance film or the like that recognizes a specific substance to be sensed to react therewith is formed on a front surface of a metal electrode (excitation electrode) provided on a quartz-crystal piece. When the adsorption layer is brought into contact with the sample solution, the adsorption layer reacts with the substance to be sensed contained in the sample solution to adsorb the substance to be sensed, resulting in a mass change in the adsorption layer. Since a natural frequency of the quartz-crystal resonator changes according to the mass change of the adsorption layer, the concentration of the substance to be sensed is measured by using this action. As the biological substance film, a film of an antibody reacting with a specific antigen (substance to be sensed) is used, for instance, and this film of the antibody adsorbs the antigen.
A patent document 1 proposes a flow-cell sensor using a quartz-crystal resonator. In this sensor, the quartz-crystal resonator in which an electrode is formed is provided via silicon rubber between a support substrate and a cover having a solution inflow channel and a solution discharge channel. In the silicon rubber, a hole portion for storing a solution is formed, and a flow cell is formed by the cover, the quartz-crystal resonator, and the silicon rubber, and the solution supplied from the inflow channel is discharged from an outflow side after flowing onto the electrode of the quartz-crystal resonator to come into contact with the electrode. Being capable of continuous supply of a liquid, such a flow-cell sensor has advantages that a frequency characteristic can be easily stabilized, the liquid can be smoothly replaced, and only a small amount of a sample solution is necessary. Further, for forming an adsorption layer in this quartz-crystal sensor, a solution containing an adsorption substance is injected into the quartz-crystal sensor, whereby the adsorption substance is adsorbed by a front surface of the metal electrode.
Incidentally, the frequency change is caused also by the adhesion of a substance other than the substance to be sensed in the sample solution, for example, the adhesion of an unintended antigen or the like, to the quartz-crystal resonator or by the viscosity of the sample solution. Therefore, to enhance detection accuracy of a trace amount of the substance, it is necessary to take a measure for eliminating a frequency change caused by a disturbance such as the adhesion of the unintended substance. Here, as a method of reducing the influence of a disturbance such as a temperature change in the quartz-crystal sensor, a structure of a patent document 2 has been proposed. In this method, there is proposed a structure in which a first quartz-crystal resonator and a second quartz-crystal resonator are formed by using a common quartz-crystal piece, an adsorption substance is made to be adsorbed by an excitation electrode of one of the quartz-crystal resonators to form an adsorption layer, and the other quartz-crystal resonator is used as a reference, whereby a frequency change accompanying the temperature change is cancelled.
For higher accuracy detection, it is necessary to make an area in the excitation electrode of one of the quartz-crystal resonators, where no adsorption substance is adsorbed, and a front surface of an excitation electrode of the reference quartz-crystal resonator adsorb a blocking substance. This blocking substance is made of a component that does not adsorb a substance such as, for example, a protein, and the reason why the electrode is made to adsorb the blocking substance is to prevent the substance to be sensed from being adsorbed by the area in the front surface of the electrode, where no adsorption layer is adsorbed, thereby forming an environment where the substance to be sensed is adsorbed only by the adsorption substance and to prevent the adhesion of components other than the substance to be sensed, thereby ensuring high accuracy regarding a correspondence relation between an amount of the substance to be sensed captured by the adsorption substance and the frequency.
In the fabrication of the sensor including the two quartz-crystal resonators, it is necessary to make the excitation electrode of one of the quartz-crystal resonators adsorb the adsorption substance and the blocking substance and make the excitation electrode of the other quartz-crystal resonator adsorb only the blocking substance. However, when the flow-cell method is adopted in the structure where the first quartz-crystal resonator and the second quartz-crystal resonator are formed on the common quartz-crystal piece, the use of the aforesaid method of injecting the solution containing the adsorption substance into the quartz-crystal sensor and then injecting the solution containing the blocking substance into the quartz-crystal sensor results in the adsorption of the adsorption substance and the blocking substance by the excitation electrodes of the both quartz-crystal resonators.
To solve this, the following method is in practice. After the excitation electrode of the reference quartz-crystal resonator is made to adsorb the blocking substance in advance, the reference quartz-crystal resonator is mounted in the quartz-crystal sensor, then the solution containing the adsorption substance and the solution containing the blocking substance are sequentially injected into the quartz-crystal sensor, and the excitation electrode of the one quartz-crystal resonator is made to adsorb the adsorption substance and the blocking substance. At this time, the solution containing the adsorption substance and the solution containing the blocking substance are sequentially supplied also to the reference quartz-crystal resonator, but since the blocking substance is formed in advance on the excitation electrode of this quartz-crystal resonator, the blocking substance prevents the adsorption of the adsorption substance. This method, however, has problems that an operator has to take an increased trouble due to the need for the pre-process of making the reference quartz-crystal resonator adsorb the blocking substance in advance and the measurement time including the pre-process increases.    [Patent document 1] Japanese Patent Application Laid-open No. Hei 11-183479    [Patent document 2] Japanese Patent Application Laid-open No. 2007-108170